Human oocytes and embryos must be held as closely as possible at a stable 37 degree C.

Temperature also affects the pH value of the sample.

Evaporation affects the pH value of the sample.

A
significant, but poorly-recognized, confounding aspect of temperature
control during the microscopic observation of oocytes and embryos in
dishes is that the design of all disposable plastic dishes does not
allow the base of the dish to come into direct contact with the
microscope stage, there is always an air gap (see Figure). Because air
is a poor conductor of heat, this air gap greatly reduces the efficacy
of heated stages (either those attached to or built into the microscope,
or the heated work surfaces of the cabinet, allowing the medium in
dishes to cool below the temperature at which the heated surface is set.

B. Culture Medium Osmolarity and pH

The
composition of culture media used for human IVF have evolved markedly
over recent years with the increasingly widespread use of "sequential
culture media" suites that comprise a range of media and buffers
designed to provide optimized support to each stage of the process from
oocyte retrieval through fertilization and embryo cleavage to the
blastocyst.

Correct use of all culture media requires the use of special atmospheres that are, at a minimum, enriched in carbon dioxide.

Additionally
it is now recognized that the human embryo shows better development,
and implantation potential, if the oxygen tension is reduced from that
in normal air. However, there is great confusion between CO2 and O2
concentrations (expressed in volumetric percentage terms) and their
partial pressure. Firstly, at higher elevations, not only does
atmospheric pressure decrease, but the relative proportions of oxygen
and nitrogen (and the other minor gases) also change: for example,
whereas there is 20.95% O2 at sea level, at an elevation of 1000 m there
is only 18.55% O2 and at 1600 m (e.g. Denver) there is just 17.2%. The
same changes affect CO2, so that if one wishes to achieve a pH of 7.3 in
a solution of 25mM bicarbonate at sea level, 6.0% CO2 is required
according to the Henderson-Hasselbach equation otherwise the pH will
shift and bicarbonate ions will be lost until the solution reaches a new
equilibrium. But at 1600 m above sea level, almost 7.5% CO2 is required
to achieve the correct partial pressure (ppCO2) to maintain the
bicarbonate ions in solution and the pH at 7.3.

A major problem
with bicarbonate-buffered media is that they take a long time to reach
equilibrium, but out-gas very quickly. Recent studies [4] have shown
that a 50 ul droplet of medium under oil will out-gas after removal from
a CO2 incubator so that the pH has shifted above 7.45 within 2 minutes
and that after replacing the dish in the CO2 incubator it will take 35
minutes to re-equilibrate the pH (only about 15 min for Petri dishes
containing 5ml of medium). These differences are due to the relative
magnitudes of the differential CO2 contents between the equilibrated
medium and air and between the incubator's atmosphere and the partially
out-gassed medium.

Finally in this regard, if culture medium is
exposed to air that is not well-saturated with water vapor then there
will be evaporative loss from the medium with concomitant increases in
medium osmolarity. Evaporation is also higher at warmer temperatures.
Whereas culture under oil helps combat this problem, during oocyte
retrievals the dishes are typically open without an oil overlay (which
would greatly complicate the procedure).

C. Air Quality

Because
of their high metabolic and cell division rates, embryos are highly
sensitive to toxic chemicals, thereby making IVF/ART laboratories and
procedure rooms especially high risk areas for volatile organic
compounds (VOCs) and other air pollutants.

It is a common
misconception that HEPA (high efficiency particulate air) filtration
removes gaseous organic and inorganic molecules. HEPA, as its name
implies, is a highly efficient system for removing particulates from the
air (the standard requires a 99.97% efficiency for a 0.3 um particle),
not low molecular weight gaseous molecules.